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Regulation of Diacylglycerol Acyltransferase-2 and Triacylglycerol Synthesis by Protein Ubiquitination



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Triacylglycerol is the major source of stored energy in humans, yet, excessive accumulation of triacylglycerols in tissues leads to obesity, diabetes and heart disease. Triacylglycerol synthesis is catalyzed by diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2. Although recent studies have shed light on the metabolic functions of these enzymes, little is known about their regulation. We have found that DGAT2 is a short-lived protein and is degraded via the ubiquitin- proteasome pathway. Our objective was to identify the lysine residues that are ubiquitinated and determine the role of ubiquitination in regulating DGAT2 stability and triacylglycerol synthesis. Initial experiments found that a lysine-less DGAT2 mutant (Lys-less-DGAT2) was not degraded. Moreover, Lys-less-DGAT2 exhibited altered subcellular localization and disrupted lipid droplet biogenesis. Screening of a DGAT2 lysine-to-arginine mutant library demonstrated that several lysine residues are involved in regulating DGAT2 stability. Substitution of two lysine clusters was sufficient to mislocalize DGAT2 and perturb typical lipid droplet formation, suggesting that these lysines may have a role in targeting DGAT2 to lipid droplets. Interestingly, DGAT2 on lipid droplets was ubiquitinated and stimulating lipogenesis did not reduce DGAT2 degradation. Monoacylglycerol acyltransferase (MGAT) enzymes, MGAT2 and MGAT3, are closely related to DGA T2 and produce the DGA T2 substrate, diacylglycerol. MGA T2 and MGA T3 interact with DGAT2 and appear to stabilize it. We determined that DGAT2, MGAT2 and MGAT3 are targeted for endoplasmic-reticulum-associated degradation (ERAD), as ERAD inhibition caused poly-ubiquitinated species of all three proteins to accumulate. Moreover, overexpression of DGAT2 and MGAT2 resulted in redistribution of the ERAD ATPase, valosin-containing protein (VCP/p97), where it becomes concentrated in the ER, co-localizing with both DGAT2 and MGAT2. Interaction of DGAT2 and MGAT2 with VCP/p97 was also demonstrated in situ. We took a non-targeted mass spectrometry approach to identify proteins interacting with DGAT2. The carbohydrate binding protein, calnexin, was one of the candidates identified. This interaction was confirmed in vitro and in situ. The possible impacts of calnexin on triacylglycerol metabolism were examined in calnexin knockout mouse embryonic fibroblasts, which exhibited stunted lipid droplet size compared to wild-type cells. Collectively, these investigations provide insight into the post-translational regulatory mechanisms of DGAT2 and DGAT2 family members.



Triacylglycerol: DGAT2



Doctor of Philosophy (Ph.D.)






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